Modified Project Summary/Abstract Section Our research on polyketide assembly lines is helping bring about a paradigm shift for how sets of component enzymes cooperate to biosynthesize polyketide natural products. The updated definition of a module, with the ketosynthase domain positioned at its downstream end, affects every level of modular polyketide synthase enzymology. Each of these levels must be further explored to achieve our long-term goal of reprogramming polyketide assembly lines to synthesize designer molecules and accelerate the drug discovery process. We first will study how ketosynthases gatekeep such that only one type of polyketide intermediate is selected by a module to be further elongated by the downstream assembly line (Specific Aim 1). This will be accomplished through methodology we have developed to crystallographically observe polyketides bound in ketosynthase active sites and measure the activity of ketosynthases mutated at suspected gatekeeping residues. We next propose to make platforms for enzymological, engineering, and structural biology studies through the reconstitution of short natural assembly lines and the engineering of short model assembly lines (Specific Aim 2). By comparing hybrid synthases constructed using the traditional module boundary with those constructed using the updated one, the use of the updated boundary could be validated through rate measurements and product characterization. With these simplified synthases, the enzymes and docking domains of the module can be studied with greater biochemical rigor. Model synthases actively synthesizing polyketides will also be examined by cryo-electron microscopy. Our lab has collected several pieces of structural evidence for higher-order architecture. In the bacillaene polyketide synthase, a three-helix element adjacent to the ketosynthase domain seems to zipper homodimeric assembly lines into ~100 MDa assembly sheets observable within Bacillus subtilis cells. We propose to understand the structures of such biosynthetic megacomplexes by reconstituting them in vitro and observing them through electron microscopy (Specific Aim 3). Our lab is already visualizing Pks12 from Mycobacterium tuberculosis both in its “bimodular” and its polymeric assembly line states. We seek to determine how modules stack to construct higher-order architecture in such systems. Through investigations at each of these levels, an overall picture of the architectures and activities of polyketide assembly lines will emerge that will be particularly significant to the future engineering of these medicinally-relevant molecular machines.